Targeted Overexpression of IGF-I Evokes Distinct Patterns of Organ Remodeling
in Smooth Muscle Cell Tissue Beds of Transgenic Mice
Jianwei Wang,* Wen Niu,* Yuri Nikiforov,* Shinji Naito,* Steven Chernausek,‡ David Witte,§ Derek LeRoith,i
Arthur Strauch,¶ and James A. Fagin*
*Division of Endocrinology and Metabolism, ‡Division of Pediatric Endocrinology, §Division of Pediatric Pathology, University of
Cincinnati, Cincinnati, Ohio 45267; iDiabetes Branch, National Institutes of Diabetes and Digestive and Kidney Diseases, National
Institutes of Health, Bethesda, Maryland 20892; and ¶Department of Cell Biology, Neurobiology and Anatomy, Ohio State University,
Columbus, Ohio 43210
Abstract
Smooth muscle cells (SMC) of the vascular wall, bladder,
myometrium, and gastrointestinal and respiratory tracts retain the ability to proliferate postnatally, which enables
adaptive responses to injury, hormonal, or mechanical stimulation. SMC growth is regulated by a number of mesenchymal growth factors, including insulin-like growth factor I
(IGF-I). To explore the function of IGF-I on SMC in vivo,
the mouse SMC a–actin promoter fragment SMP8 (21074
bp, 63 bp of 59UT and 2.5 kb of intron 1) was cloned upstream of rat IGF-I cDNA, and the fusion gene microinjected to fertilized eggs of the FVB-N mouse strain. Mating
of hemizygous mice with controls produced about 50%
transgenic offspring, with equal sex distribution. Transgenic
IGF-I mRNA expression was confined to SMC-containing
tissues, with the following hierarchy: bladder . stomach .
aorta 5 uterus . intestine. There was no transgene expression in skeletal muscle, heart, or liver. Radioimmunoassayable IGF-I content was increased by 3.5- to 4-fold in aorta,
and by almost 10-fold in bladder of transgenic mice at 5 and
10 wk, with no change in plasma IGF-I levels. Wet weight
of bladder, stomach, intestine, uterus, and aorta was selectively increased, with no change in total body or carcass
weight of transgenic animals. In situ hybridization showed
that transgene expression was exquisitely targeted to the
smooth muscle layers of the arteries, veins, bladder, ureter,
stomach, intestine, and uterus. Paracrine overproduction of
IGF-I resulted in hyperplasia of the muscular layers of
these tissues, manifesting in remarkably different phenotypes in the various SMC beds. Whereas the muscular layer
of the bladder and stomach exhibited a concentric thickening, the SMC of the intestine and uterus grew in a longitudinal fashion, resulting in a marked lengthening of the small
bowel and of the uterine horns. This report describes the
first successful targeting of expression of any functional
protein capable of modifying the phenotype of SMC in
transgenic mice. IGF-I stimulates SMC hyperplasia, leading to distinct patterns of organ remodeling in the different
Address correspondence to James A. Fagin, M.D., Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, 231 Bethesda Ave., Room 5564, Cincinnati, OH 45267-0547.
Phone: 513-558-4444; FAX: 513-558-8581; E-mail: [email protected]
Received for publication 24 March 1997 and accepted in revised
form 30 June 1997.
The Journal of Clinical Investigation
Volume 100, Number 6, September 1997, 1425–1439
http://www.jci.org
tissue environments. (J. Clin. Invest. 1997. 100:1425–1439.)
Key words: alpha actin • IGF binding proteins • hyperplasia •
small intestine • bladder
Introduction
Smooth muscle cells (SMC)1 provide structure, elasticity, and
contractile properties to a number of tissue compartments, including the peripheral blood vessels. SMC of the vascular wall,
urinary bladder, myometrium, and gastrointestinal and respiratory tracts retain the capability to proliferate postnatally,
which enables a number of adaptive responses to injury, hormonal or mechanical stimulation. Growth of SMC is subject to
regulation by a number of mesenchymal growth factors, including insulin-like growth factor I (IGF-I).
IGF-I, a small polypeptide with structural homology to
IGF-II and proinsulin, is produced by many cell types and acts
as an autocrine/paracrine growth factor. It also functions as a
circulating hormone, as it is present at high concentrations in
plasma, where it is associated with specific binding proteins
(IGFBP). The relative contribution of locally produced and
circulating IGF-I to the biological activity of the growth factor
remains unclear. This is complicated by the fact that each tissue environment has a specific set of IGFBPs and IGFBP-specific proteases that modulate the bioavailability of IGF-I and
its access to its specific cognate membrane receptor. There is
ample evidence that IGF-I stimulates SMC growth in vitro (1,
2). Information on the possible action of IGF-I in SMC tissue
beds in vivo, however, is conjectural, and based primarily on
descriptions of the regulation of IGF-I gene expression in association with events that trigger smooth muscle hyperplasia. After balloon arterial denudation, there is a marked induction of
IGF-I gene expression in the medial layer of the rat aorta, coincident with the peak time of SMC DNA synthesis (3–5). Estrogen and progesterone induce IGF-I and IGF-II mRNA levels in myometrial cells, consistent with the notion that these
growth factors may help mediate sex-steroid–dependent SMC
proliferation in the uterus (6–8). Bladder hypertrophy secondary to partial urethral ligation is also associated with increased
IGF-I biosynthesis (9). It is unclear whether the local expression of IGF-I is critical to the development of smooth muscle
hyperplasia in these tissue environments, or if it is simply a
passenger event.
The object of the experiments described here was to examine the function of IGF-I in SMC by targeting its expression to
the appropriate cells of transgenic mice. To select an appropri-
1. Abbreviations used in this paper: CAT, cloramphericol acetyl transferase; GH, growth hormone; SM, smooth muscle; SM-MHc, smooth
muscle myosin heavy chain; SMC, smooth muscle cells.
IGF-I Overexpression in Smooth Muscle Cells of Transgenic Mice
1425
ate promoter capable of delivering SMC-specific expression,
we considered information on the pattern of expression of several genes during vascular and intestinal development. Perhaps the most extensively studied of these is the smooth muscle (SM)-a actin gene, the expression of which is primarily
restricted to SMC of adult rodents and rabbits (10–12). This
gene is a member of the actin multigene family that consists of
three classes of isoforms in vertebrates, named cytoplasmic,
striated, and smooth muscle, based on the distribution of expression in adult animals. During early stages of embryogenesis, SM-a actin is also expressed in skeletal and cardiac muscle,
and only becomes restricted to SMC during late fetal maturation (10, 13, 14). By contrast, smooth muscle myosin heavy
chain (SM-MHc) is thought to be a highly specific marker of
the SMC lineage even during neonatal life, as it is expressed
exclusively in the vasculature, intestine, lung, stomach, and
uterus of the developing and the adult mice (15). However,
preliminary evidence suggests that the activity of a SM-MHc
promoter fragment coupled to luciferase is quite weak in transgenic mice (16). SM22a, a putative calcium binding protein, is
expressed in cardiac, smooth, and skeletal muscle during embryogenesis (17, 18). A fragment of the SM22a promoter directs reporter expression that is confined to arterial, but not
venous or visceral smooth muscle cells. We elected to target
IGF-I through mouse SM-a actin promoter constructs, primarily because of the expectation that their activity would manifest in most if not all SMC beds of adult animals, and thus allow us to examine the role of IGF-I on these cells in various
tissue contexts. To our knowledge, this is the first report of
overexpression of a functional protein capable of modifying
the phenotype of SMC in transgenic mice. Our data shows that
the SMP8–IGF-I transgene directs expression uniquely to
SMC, where it promotes hyperplasia without affecting plasma
IGF-I levels or total body weight. Quite unexpectedly, the pattern of organ remodeling evoked by IGF-I was distinct in the
different smooth muscle tissue environments, resulting in concentric thickening of the muscular layers of the bladder and
stomach, and longitudinal growth of the uterus and intestine.
Methods
Construction of SM-a actin/IGF-I and SM-a actin/cloramphericol
acetyl transferase (CAT) fusion genes. The SMP8–IGF-I chimeric gene
was constructed by fusing a fragment of the mouse SM-a actin 59 end
(SMP8) to a rat IGF-I cDNA. SMP8 consists of 21074 bp of the 59
flanking region, the 63-bp 59UT, and about 2.5 kb of the first intron
(19–21). A 0.67-kb PvuII–BspM1 fragment from pIGF-I was cloned
into pBluescript SK (2) at the EcoRv site. The resulting plasmid,
prIGF-I, was cut with EcoR1, filled in with Klenow, and then restricted with BamH1 to release rat IGF-I cDNA (rIGF-I) containing
the entire coding sequence, 57 bp of 59-UT derived from exon 1 of the
gene and 95 bp 39-UT (22). A 0.24-kb Sau3 A1 fragment of the SV40
early polyadenylation signal (SV40-pA) was cloned into pBluescript
SK (2) at the Pst1 site, and the resulting plasmid, pSV40-pA, was cut
with Sma1 and EcoR1 to release the SV40-pA fragment. The rIGF-I
and SV40-pA fragments were cloned into BamH1-EcoR1–digested
pSMP8 by triple ligation to create pSMP8–IGF-I. pSMP8-CAT consisted of the indicated 3.6-kb promoter insert cloned into the Sph1–
BamH1 polylinker site of pBLCAT3, upstream of the CAT reporter
(23). The diagrams of the pSMP8–IGF-I and pSMP8-CAT constructs
are shown in Fig. 1.
Generation of transgenic mice. The SMP8–IGF-I fusion gene was
excised from pSMP8–IGF-I by restriction with SphI and EcoR1,
whereas pSMP8-CAT was linearized with SphI before microinjection. The product was size separated and isolated from the agarose
gel using the Geneclean II kit (Bio 101, La Jolla, CA). The DNA was
sequentially extracted in phenol, chloroform, and ether, and then dialyzed against 5-mM Tris, pH 7.4, and 0.1-mM EDTA. The male pronuclei of fertilized eggs from either C3HEB/FEJ (for SMP8-CAT) or
FVB-N (for SMP8–IGF-I) mouse strains were microinjected with 2 pl
of linearized DNA at the transgenic mouse facility of the University
of Cincinnati, Cincinnati, OH. Microinjected eggs were implanted
into the oviduct of pseudopregnant female mice and carried to term.
Figure 1. Linear map of the SMP8IGF-I and SMP8-CAT fusion genes.
SMP8–IGF-I: the mouse SM-a actin promoter consisted of 21074 bp
of 59 flanking region (light gray
box), the transcription start site,
48 bp of exon 1 (black box), the
2.5-kb intron 1 (line), and the 15-bp
exon 2 (black box) of SM-a actin
fused to a 0.7-kb rat IGF-I cDNA
(dark gray box), followed by a
240-bp SV40 early polyadenylation
signal sequence (open box). SMP8CAT: The SM-a actin promoter
was cloned upstream of the
chloramphenicol acetyltransferase
reporter cDNA (dark gray box) in
pBLCAT3 (23), the small T intron
(line), and the SV40 polyadenylation signals (open box). The following riboprobes were used for in situ
hybridization: (a) complementary
to rat IGF-I mRNA cross-hybridizes with mouse IGF-I mRNA;
(b) complementary to SV40 poly
(A) signal sequence (specific for
transgene).
1426
Wang et al.
Positive founders for SMP8-CAT or SMP8–IGF-I were identified by
Southern blotting, and bred to wild-type C3HEB/FEJ or FVB mice,
respectively. Transgene copy number was calculated by quantitative
Southern blotting, using known amounts of the SMP8–IGF-I construct added to nontransgenic mouse genomic DNA as a standard according to the following formula: 7,200 bp (size of SMP8–IGF-I) 3 5
mg / 6 3 109 bp/copy 5 6 3 1026 mg/copy 5 6 pg/copy.
Screening of transgenic mice. Heterozygotes and nontransgenic
littermates from the F1 and subsequent generations were selected by
Southern blotting of genomic DNA. Mouse tail tips (z 1 cm) were
cut at 3 wk of age, and digested with proteinase K (100 mg/ml) in 0.5
ml of 10-mM Tris, pH 8.0, 100-mM EDTA, 0.5% SDS at 508C overnight. After phenol/chloroform/isoamyl alcohol (25:24:1, pH 8.0) extraction, the high molecular mass DNA was precipitated in isopropanol, washed in 70% ethanol, dissolved in ddH2O and stored at 48C
until use. Southern analysis was performed as described (24). Briefly,
5 mg of mouse genomic DNA was digested with HindIII at 378C overnight, fractionated on a 1% agarose gel, transferred to a Hybond-N
nylon membrane (Amersham Corp., Arlington Heights, IL), and
then hybridized with a 0.7-kb HindIII-XhoI fragment of mouse SMa–actin genomic DNA labeled by random priming (Prime-It II kit;
Stratagene, Inc., La Jolla, CA). Endogenous mouse SM-a actin alleles gave bands of 2.5 kb, whereas the transgene was identified as an
z 5-kb band.
RNA isolation and Northern blot analysis. Total RNA was isolated from tissues by a single step acid guanidinium thiocyanate-phenol-chloroform extraction method. Northern blots were performed as
described (25). Briefly, 5 mg of tissue total RNA was gel-separated,
transferred to a nylon membrane, and then hybridized with random
primed rat IGF-I cDNA. For standardization, blots were rehybridized with either human glyceraldehyde-3-phosphate dehydrogenase
cDNA, when comparing within the same tissue type, or 18S rRNA,
for comparisons between different tissues.
Allometry of SMP8–IGF-I transgenic mice. Transgenic mice and
their nontransgenic littermates were killed by CO2 asphyxiation. After obtaining body weight, blood was collected by cardiac puncture
and the serum stored at 2808C until use. Organs of interest, except
the aorta, were dissected, rinsed in ice-cold PBS, tissue-blotted,
weighed, and immediately frozen in dry ice. Contents of stomach and
small intestine were flushed out with PBS before weighing. A section
of the arterial vessel, from the aortic arch to the level of femoral fork,
was excised and placed in PBS. Adhering fat and connective tissue
from the adventitia were scraped off under surgical microscope and
the vessel was cleansed with a copious amount of PBS to remove any
residual blood. Organs were immediately placed on dry ice and
stored at 2808C until use.
Tissue extraction and RIA for IGF-I. Tissues were placed in 1-ml
ice-cold 1-M acetic acid and immediately homogenized for either 30 s
(for bladder) or 1 min (for aorta) on ice with a Polytron PT3000 at
full speed. After standing on ice for a further 2 h, the tissue extracts
were centrifuged in siliconized Eppendorf tubes at 18,000 g for 1 h at
48C. The supernatants were carefully removed and aliquots were pipetted into polystyrene tubes to evaporate the solvent in a SpeedVac
device (Savant Instruments, Inc., Farmingdale, NY) for 1 h without
heating. Small aliquots of tissue extracts were also saved for measurement of total protein. Protein content was determined by the dyebinding method using BSA as a standard (26).
Recombinant human IGF-I (Austral Biologicals, San Ramon,
CA) was iodinated according to the method described by Hill et al.
(27). Briefly, 1 mg of IGF-I in 0.1% HAc was iodinated with 1 mCi
[125I]NaI by addition of chloramine T (0.1 mg/ml in 0.3-M sodium
phosphate buffer, pH 7.6) in a stepwise manner over a period of 4.5
min. The iodination reaction was terminated by sequentially adding a
saturated tyrosine solution and 60-mM NaI to the reaction mixture,
which was then applied to a pre-equilibrated Sephadex G-50 column
and eluted with 4-mM HCl, 75-mM NaCl, and 0.1% BSA to separate
the iodinated peptide from the free 125I. The specific activity of the
purified [125I]iodyltyrosyl–IGF-I was estimated at 320 mCi/mg.
For the IGF-I RIA, tissue extracts were resuspended in 0.03-M
sodium phosphate (pH 7.4) containing 0.05% Tween 20, 0.02% NaN3,
0.02% protamine sulfate, and 0.01-M EDTA, and assayed using a
double antibody precipitation, nonequilibrium assay as described
previously (28). The IGF-I antiserum was provided by Dr. L.E. Underwood (Pediatric Endocrinology, Chapel Hill, NC), and recombinant human IGF-I used as a standard. Tissue extracts and rodent sera
gave parallel displacement curves as the human IGF-I standard.
Western ligand blotting. To determine tissue and serum content
of IGFBPs, frozen tissues (bladders and aortas) were homogenized in
PBS with 12-mM EDTA and 1-mM PMSF in a Polytron (model
PT3000) at full speed on ice. Extracts were centrifuged at 100,000 g
for 1 h at 48C, and the supernatant further concentrated in a Centricon-3 device (Amicon, Inc., Beverly, MA). Aliquots of the concentrated extracts were taken for protein assay. For Western ligand blotting, serum samples (8 ml each) were run on 7.5–20% gradient
Laemmli gel and tissue extracts were run on 10–20% gradient or 10%
Laemmli gels, under nonreducing conditions. After blotting, the nitrocellulose membrane was sequentially incubated with PBS-0.1%
Tween-20 and PBS-1% BSA, and then with 24 mCi [125I]IGF-1 in 150
ml of PBS containing 1% BSA-0.1% Tween-20 at 48C for 16 h. After
washing, the blots were dried and exposed to x-ray film at 2708C. For
tissue immunoblotting, the extracts were run on the same gels as used
in ligand blotting but under reduced conditions. The blots were
blocked with a TBS solution containing 0.05% Tween-20 and nonfat
dry milk and incubated with either 1:5000 rabbit anti–rat IGFBP4 antibody (516.F) or 1:1000 anti–human IGFBP-5 antibody (UBI, Lake
Placid, NY) in the same blocking solution plus 0.01% thimerosal at
room temperature for 16 h. Membranes were washed and then incubated with 1:2500 goat anti–rabbit antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) for 1 h at room temperature. The blots were detected with SuperSignal reagent (Pierce Chemical Co., Rockford, IL).
In situ hybridization. In situ hybridization was performed as described previously (29). Tissues dissected from animals at the indicated ages were fixed in 4% paraformaldehyde, saturated overnight
with 30% sucrose in PBS, and frozen in OCT. Cryostat sections (7
mm) were mounted on silane-coated slides. Sense and antisense
cRNA probes for either rat IGF-I, that hybridizes to both endogenous mouse and transgenic rat IGF-I mRNA, and for a transgenespecific SV40 39UT/polyA signal sequence, that recognizes only the
exogenous mRNA, were labeled with [35S]rUTP, using a commercially available kit (Stratagene, Inc.). For generation of the antisense
IGF-I riboprobe, the prIGF-I plasmid was linearized with BamHI,
and transcribed with T7 RNA polymerase. The product was 733 bases
long, 678 of which were complementary to transgene mRNA (see Fig.
1, probe A). The sense IGF-I riboprobe was 732 bases long, and was
obtained after linearizing the same vector with EcoRI, and transcribing with T3 RNA polymerase. Transgene-specific riboprobes were
obtained from the pSV40-pA plasmid. The antisense probe was transcribed from HindIII-linearized plasmid with T7 polymerase, generating a 351 base riboprobe, 150 bases of which were complementary
to the transgenic sequence (see Fig. 1, probe B). The 343 base sense
riboprobe was generated from BamHI-linearized pSV40-pA with T3
polymerase. Hybridization was performed with a total of 5 3 105 to
1 3 106 cpm in a final volume of 30 ml per slide. The sections were hybridized overnight at 428C, treated with 50 mg/ml RNAase A (Sigma
Chemical Co., St. Louis, MO) and 100 U/ml of RNAase T1 (Boehringer Mannheim Biochemicals, Indianapolis, IN) for 30 min at 378C,
and washed to a final stringency of 0.1 3 standard citrate saline at
508C. Slides were dipped in NTB2 emulsion (Eastman Kodak Co.,
Rochester, NY), diluted 1:1 with 0.6 mol/liter ammonium acetate, exposed for 10–14 d, and developed in D19 developer (Eastman Kodak
Co.). Sections were counterstained in hematoxilin and eosin and photographed under dark and brightfield illumination.
Tissue histomorphometry. Morphometry was done using NIH
Image V1.61, an image processing and analysis program for the Macintosh. The tissue section images of SMP8–IGF-I transgenic mice
(line 23423) and their age-matched nontransgenic controls were
IGF-I Overexpression in Smooth Muscle Cells of Transgenic Mice
1427
color-captured into the computer from the trichrome-stained sections
through the microscope. After adjusting the image contrast, the area
of interest was auto-outlined and the regions outside and inside of the
area cleared. For example, the lumen area of aorta and intestine, and
the thickness of the aortic media and the intestinal smooth muscle
were calculated from a hypothetical perfect circle. The formulas are
as follows: Do 5 P/p; Ai 5 P2/4p 2 A; Di 5 2(Ai/p)0.5 , T 5 (Do 2
Di)/2 , where Do is the outer diameter, P is the perimeter, Ai is the
lumen area, A is the measured area, Di is the inner diameter, and T is
the thickness of the muscular area.
CAT assay. Tissues from SMP8-CAT transgenic mice were dissected, rinsed in ice-cold PBS and immediately frozen in liquid N2 until
assayed. Assays for CAT activity were performed essentially as described (30, 31) using 14[C]chloramphenicol (57 mCi/mmol; Amersham Corp.) and equivalent amount of tissue extract (typically 1 mg
protein per 150 ml of reaction volume). Acetylated products were
separated by thin layer chromatography using Whatman silica gel
60A multichannel plates and exposed to Kodak XAR-5 film (Eastman Kodak Co.). TLC autoradiograms were analyzed using Image
Quant software and laser densitometry (Molecular Dynamics, Sunnyvale, CA) to estimate the conversion of 14[C]chloramphenicol to its
acetylated derivatives.
Determination of myocyte number, tissue DNA, and RNA content. RNA and DNA were extracted from bladder, intestine, stomach,
and brain tissues of SMP8–IGF-I transgenic mice and their nontransgenic littermates as described (32). The concentrations of the extracted nucleic acids were measured by UV absorption at 260 nm. To
verify RNA/DNA data, the myocyte number per surface area was directly counted on hematoxilin and eosin-stained slides using a grid as
a reference point. For each tissue examined, a minimum of five fields
(340) of five sections was counted.
Statistics. Comparisons were made using Student’s t test.
Results
The mouse SMP8 a-actin promoter targets reporter activity to
SMC-rich tissues of transgenic mice. Transcriptional activity
directed by the 21074 bp SM-a actin promoter (SMP8) has
been found to be restricted to myogenic lines in vitro (19–21).
To determine if this promoter fragment also conferred selective expression in vivo, CAT activity was measured in tissue
extracts of transgenic mice derived from a representative
founder carrying the SMP8-CAT fusion gene. Acetylation activity was highest in SMC-rich tissues (stomach, aorta, mesenteric artery), intermediate in lung and kidney, and negligible in
cardiac ventricle and liver (Fig. 2).
Generation of SMP8–IGF-I mice, and examination of transgene expression. Eight SMP8–IGF-I founder animals were obtained from a total of 42 mice screened. The percentage of
transgenic offspring in the F1 generation indicated that three
of the eight founders were mosaics. In subsequent generations,
matings of hemizygous transgenic mice with controls produced
about 50% transgenic offspring, with equal sex distribution.
Five transgenic lines were further propagated for more complete analysis.
Figure 2. Acetylation activity of
selected tissues of SMP8-CAT
transgenic mice (line 2). Data
(mean6SEM) represent the optical
density of autoradiograms of thin
layer chromatograms of the indicated tissue extracts harvested from
three mice, normalized per microgram of total protein.
1428
Wang et al.
Figure 3. (a). Northern blot analysis of SMP8-IGF-I transgene expression in tissues from a representative transgenic
mouse and its littermate. 5 mg of tissue total RNA was gelseparated, transferred to a nylon membrane, and then sequentially hybridized with a rat IGF-I cDNA (A) and human
18S probes (B). Endogenous mouse IGF-I mRNA splice
variants are apparent in liver and to a lesser extent in the
uterus (mIGF-I). The IGF-I transgene mRNA was distinguished from the endogenous forms by size (transgene), and
was expressed primarily in SMC-rich tissues. (b): SMP8IGF-I transgene expression in aorta and bladder tissues of
normal and SMP8-IGF-I transgenic mice from several lines.
5 mg of tissue total RNA was gel-separated, transferred to a
nylon membrane, and then sequentially hybridized with rat
IGF-I cDNA probe (A) and human GAPDH probe (B).
The tissue distribution of expression of endogenously produced and transgenic IGF-I mRNA is shown in Fig. 3 A. RNA
was obtained from tissues dissected from a 5-wk-old SMP8–
IGF-I transgenic female and a nontransgenic littermate. IGF-I
mRNA was abundant in the liver of the nontransgenic mouse,
and otherwise detected only in the uterus. By contrast, the
IGF-I transgene was detected at high levels in bladder, aorta,
intestine, stomach, uterus, and spleen. The size of the transgenic IGF-I mRNA was slightly greater than that of the endogenous transcript, presumably because of differences in the
composition of the 39UT and polyA region of the transgenic
message. Transgene expression in skeletal muscle and in the
heart was barely detectable. Overexpression of the exogenous
IGF-I mRNA was consistently found exclusively in SM-rich
tissues of transgenic mice derived from all founder lines tested,
although to varying degrees, as shown in Fig. 3 B. There was
no correlation between transgene copy number in the various
lines (which ranged from 2 to 20 copies per cell), and expression of IGF-I mRNA (not shown).
IGF-I content of tissue extracts was measured by RIA, after separation of IGF binding proteins by acidification. The
appropriateness of the separation was verified by demonstrating that the immunoreactive fraction in the acidified tissue
extract coeluted with recombinant IGF-I after separation
through a Sephadex G50 column. IGF-I content of aorta and
bladder was many-fold higher in SMP8–IGF-I mice as compared to their nontransgenic littermates at the two time points
tested (5.5 and 10 wk) (Fig. 4 A). There was no significant difference in plasma IGF-I levels in the SMP8–IGF-I transgenic
line f23423 (Fig. 4 B). There was a tendency towards higher
plasma IGF-I levels in the transgenic line f23988, that was not
statistically significant. As will be discussed below, line f23988
IGF-I Overexpression in Smooth Muscle Cells of Transgenic Mice
1429
Figure 4. (Left) Tissue IGF-I concentrations in aorta and bladder of
SMP8–IGF-I transgenic mice line
23423 (TG) and their nontransgenic
littermates (NT). Tissue protein
extracts were acidified to separate
them from binding proteins, and the
supernatants were subjected to
RIA. Results are expressed as
picograms of IGF-I per milligram
of total acid-solubilized protein
from individual tissues. Bars represent mean6SEM from tissues
obtained from 5- (n 5 3) and
9-wk-old (n 5 4) SMP8-IGF-I TG
and NT littermates. (Right) Serum
IGF-I concentrations in SMP8–
IGF-I transgenic mice (TG)
derived from two transgenic lines
(f23988 and f23423) and their nontransgenic littermates (NT). Binding
proteins were removed from serum
samples by acidification before
RIA. Results are expressed as
nanograms of IGF-I per milliliter of serum. Data represent n 5 4 for each TG or NT group. There was no significant difference of serum IGF-I
between TG and NT mice of either line.
was the only one displaying a modest increase in total body
and carcass weight, suggesting that the overexpression of
IGF-I in these animals was not entirely confined to paracrine
tissue environments.
Total body weight and allometry of SMP8–IGF-I transgenic
mice demonstrates selective growth of bladder, stomach, uterus,
intestine, and aorta. Total body, carcass, and individual organ
weights were determined in mice derived from two of the lines
(f23423 and f23988), at two different time points. As shown in
Fig. 5, f23423 mice had no change in total body weight compared to their nontransgenic littermates. By contrast, f23988
mice had a modest increase in total body weight that was already apparent at 4 wk of age, and which was sustained at
about 115 and 120% that of nontransgenic males, and females,
respectively. In the f23423 line there was a selective increase in
the wet weight of the aorta, bladder, intestine, and stomach in
both male and female mice. In addition, females had a marked
increase in uterine weight that was already apparent before
puberty (Table I). As mentioned above, mice from the f23988
line were the only ones to exhibit a modest increase in total
body weight. However, even in these animals the increase in
the weight of the bladder, intestine, stomach, and uterus was
markedly disproportionate to total body weight gain, as demonstrated after controlling for this variable. Mice from f23421
and f23408 exhibited similar findings (not shown).
Transgene expression localizes exclusively to smooth muscle
cells. The cellular localization of expression of IGF-I mRNA
was examined first by in situ hybridization with a rat IGF-I
cRNA, that recognized both the endogenous as well as the
transgenic gene product. As reported previously, the major
site of IGF-I gene expression in normal mice was in the parenchymal cells of the liver (Fig. 6 A). Expression of IGF-I
Figure 5. Growth curves of male (left) and female (right) SMP8–IGF-I transgenic mouse
lines f23988 and f23423 and nontransgenic
controls (NT).
1430
Wang et al.
Table I. Total Body and Tissue Weights of SMP8–IGF-I Mice and Age-matched Nontransgenic Controls
(Mean1SD)
Line
n
M
23423
IGF-I Overexpression in Smooth Muscle Cells of Transgenic Mice
9 wk
F
M
23988
15 wk
F
Body
(g)
Carcass
(g)
Aorta
(mg)
Bladder
(mg)
Brain
(mg)
Heart
(mg)
Intestine
(mg)
Kidney
(mg)
Lung
(mg)
Spleen
(mg)
Stomach
(mg)
NT
T
T/NT%
p
8
4
25.361.6
25.960.9
102.6
16.961.1
16.460.4
97.0
6.761.3
9.962.5
148.7
0.0288
13.264.1
30.3613.9
228.5
0.0152
399.3633.5
382.5613.6
95.8
124.7613.6
108.366.2
86.8
1374.5679.7
1833.4648.1
133.4
, 0.0001
468.1644.8
461.1628.7
98.5
144.3623.8
134.3617.2
93.1
92.569.9
115.1619.3
124.5
0.0338
126.0618.2
163.1611.3
129.4
0.0066
NT
T
T/NT%
p
6
4
20.160.8
20.662.4
102.4
13.360.6
12.761.7
95.9
5.861.1
9.161.1
155.6
0.0033
8.461.3
16.562.9
196.3
0.0004
401.6632.2
374.2632.8
93.2
94.763.4
92.266.1
97.3
1301.0694.7
1705.46135.3
131.1
0.0011
311.6623.8
318.1643.4
102.1
120.369.4
124.9618.9
103.8
92.865.9
78.2635.7
84.3
117.2611.5
152.1621.2
129.7
0.0170
NT
T
T/NT%
p
17
7
29.862.1
35.662.0
119.6
0.0002
20.361.5
23.161.3
113.6
0.0063
8.961.7
10.863.5
122.3
24.2610.6
54.7619.1
225.8
0.0019
438.1630.6
411.8645.2
94.0
139.4613.3
150.0612.3
107.6
1433.66129.1
2334.86198.2
162.9
, 0.0001
592.5676.5
631.0665.5
106.5
155.6616.5
165.1616.6
106.1
111.2632.1
139.6633.3
125.5
144.3613.6
248.0618.5
171.9
, 0.0001
NT
T
T/NT%
p
9
3
23.061.7
27.961.8
121.2
0.0029
15.561.0
17.761.3
114.4
0.0200
7.461.4
8.260.9
110.5
10.762.7
19.261.4
179.7
0.0007
419.1627.6
434.3632.3
103.6
107.368.1
112.065.4
104.4
1329.1692.8
2383.4655.0
179.3
, 0.0001
376.9639.5
396.0611.1
105.1
143.3621.2
160.2615.1
111.8
97.169.1
125.8611.8
129.6
0.0026
140.6610.9
247.7618.0
176.3
, 0.0001
NT, nontransgenic; T, SMP8–IGF-I transgenic. Wet weight of tissue was performed as described in Methods.
Uterus
(mg)
68.6620.6
124.9614.3
182.1
0.0028
81.4613.2
178.364.4
219.2
, 0.0001
1431
mRNA in normal uterus, stomach, and intestine of nontransgenic mice was primarily confined to the epithelial layer (Fig. 6
C), with only modest levels present in smooth muscle cells.
Similarly, IGF-I mRNA levels were very low in arterial
smooth muscle cells (not shown, and reference 3). In marked
contrast, transgenic IGF-I mRNA was very abundantly expressed in a selective fashion in the SMC compartments of all
tissues examined. The riboprobe used was complementary to a
unique sequence present only in the transgene (the SV40
polyA signal, see Fig. 1), and hybridization was only seen in
Figure 6. In situ hybridization of SMP8–IGF-I mice and nontransgenic controls. (A) Liver, nontransgenic; IGF-I riboprobe, endogenous expression is diffuse in hepatocytes. (B) Bladder, nontransgenic, IGF-I riboprobe, no significant endogenous expression in either the mucosa or muscle
layers. (C) Stomach, nontransgenic; showing junction between forestomach (right half) and gastric glandular mucosa. There is a weak signal
(arrow) in the lamina propria of the foregut mucosa. No signal in the muscular layers of the wall of the stomach. (D) Coronary artery, SMP8–
IGF-I; SV40 probe, arrow showing signal in the wall of the artery, none in surrounding cardiac muscle. (E) Bladder, SMP8–IGF-I; SV40 probe,
strong signal localized to the SMC of the bladder wall, no signal in the mucosa. (F) Uterus, SMP8–IGF-I, SV40 probe, most signal localizes to
smooth muscle cells in the wall of the uterus (arrow). Scattered positive cells in stroma. No signal in epithelial layer.
1432
Wang et al.
Figure 7. Expression of the IGF-I transgene in SMP8–IGF-I transgenic mice. (A) Section of small intestine from a transgenic mouse line that has
been hybridized with the antisense probe for SV40. There is strong signal (arrows) that localizes to the smooth muscle cells in the wall of the
bowel. (B) Section of small intestine from a nontransgenic mouse which was also hybridized with the SV40 probe. No signal is detected in the
nontransgenic animals. The yellow–green color in the crypts of the mucosal glands in both the transgenic and nontransgenic tissue is autofluorescence from the Paneth cell granules and does not represent hybridization signal. (C) Longitudinal section through the proximal bronchus of a
transgenic mouse. The SV40 signal (arrows) localizes to the SM cell layers in the wall of the bronchus. (D) Longitudinal section through the
aorta (ao) of a transgenic mouse with strong SV40 signal (arrow) in the wall of the aorta. (E) Section of renal cortex from a transgenic mouse.
The SV40 signal (arrow) localizes to the region of the juxtaglomerular apparatus but not in the glomerulus. (F) Bright field image of the kidney
with signal (arrows, black grains) that localize to the juxtaglomerular apparatus but not the glomerulus (gl). (A–E, darkfield; F, bright field;
counterstain hematoxylin and eosin; A–D 3100, E 3400, F 3600).
IGF-I Overexpression in Smooth Muscle Cells of Transgenic Mice
1433
tissues from transgenic animals. Transgene expression was observed in the media of all arterial beds examined. There was
high level expression in the aortic media (Fig. 7 D). Fig. 6 D
shows a longitudinal section of a coronary artery with a strong
signal along the margin of the vessel. Notably, there was no hybridization in the surrounding myocardial cells. Similarly,
there was no transgene localization to skeletal myocytes.
Smooth muscle cells of the bladder, uterus and intestine also
showed marked hybridization to the transgene-specific riboprobe (Figs. 6, E and F, and 7 A). Bronchial SMC also demonstrated expression of the transgene (Fig. 7 C). Interestingly,
there was no expression in glomerular mesangial cells, however, there was a clear signal in the juxtaglomerular apparatus
(Fig. 7, E and F).
Tissue morphometry reveals distinct patterns of organ remodeling. The localization of the IGF-I transgene uniquely to
SMC, coupled to the evidence that tissues rich in SMC exhibited selective weight gain, prompted a more detailed investigation of the anatomical and histological characteristics of the affected organs. The urinary bladder had the most dramatic
weight changes (Table I), and was also the site of highest transgene expression in all lines tested. As shown in Fig. 8 A, the
major feature of the transgenic bladders was a marked increase in the thickness of the muscular layer. This change was
attributable to increased cell mass, and not to a greater deposition of extracellular matrix, as demonstrated by trichrome
staining (not shown). The muscular layer of the stomach was
also more cellular and markedly thicker in the SMP8–IGF-I
transgenic animals (not shown).
The uterine phenotype of the SMP8–IGF-I transgenic mice
was in marked contrast to that of the bladder. Gross anatomical examination revealed a striking increase in the length of
the uterine horns (Fig. 8 B). Histological and morphometric
evaluation indicated that the surface area of the myometrial
layer in transverse sections was not significantly different from
that of nontransgenic littermates. Thus, SMC growth in the
uterus manifested entirely in an increase in the overall length,
but not the width, of the myometrium. This phenomenon was
also apparent in the small intestine. As shown in Fig. 8 C and
Table II, the small bowel of SMP8–IGF-I transgenic mice was
about 23% longer than that of nontransgenic littermates. Although the surface area of the muscularis layer in transverse
sections was also modestly increased, this clearly does not explain the major increase in intestinal weight and length in the
transgenic animals (Table II). Interestingly, the increase in intestinal length occurred entirely postnatally: intestinal length
was 8.360.7 cm in SMP8–IGF-I transgenics 1 d after birth (n 5
5), and 8.960.6 cm in nontransgenic littermates (n 5 4).
The effect on medial aortic smooth muscle cells was assessed by histomorphometry of transverse sections taken at
identical sites from aortas of SMP8–IGF-I transgenic mice and
their littermates (n 5 5). Overall aortic medial area and thickness were significantly greater in the IGF-I overexpressing animals compared to their littermates (Table II).
Overexpression of the IGF-I transgene is associated with
smooth muscle hyperplasia. The increase in SMC mass in
SMP8–IGF-I transgenic mice was due to hyperplasia, and not
hypertrophy, as determined by RNA/DNA and protein/DNA
ratios in the targeted tissues. As shown in Table III, there was
no difference in these parameters in the bladder, uterus, or intestine of transgenic animals. As these tissues contain a variable admixture of SMC with other cell types, the presence of
hyperplasia was also confirmed by counting myocytes on hematoxilin and eosin-stained slides of transverse sections of
bladder and aorta at a magnification of 40. Myocyte counts per
surface area were not significantly different after examining
five fields from five sections of each of these tissues from four
separate SMP8–IGF-I transgenic and nontransgenic littermates (not shown), indicating that the average size of the
smooth muscle cells was not different between transgenics and
controls.
Expression of IGF binding proteins in SMP8GF-I transgenic mice. The biological activity of IGF-I is modified by locally produced IGFBPs, that vary in their expression and
Table II. Morphometry of Aorta and Intestine from SMP8–IGF-I Transgenic Mice and their Nontransgenic Controls
(Mean1SD)
Aorta
NT
T
T/NT%
P
Medial area
(mm)2
Outer perimeter
(mm)
Lumen area
(mm)2
Medial thickness
(mm)
6243168897
7679262551
123.0
0.0146
22066142
23346125
105.8
326625641004
358256646750
109.7
2963
3562
117.6
0.0113
(Mean1SD)
Intestine
NT
T
T/NT%
P
Length
(cm)
Muscular area
(mm)2
Muscular perimeter
(mm)
Muscular thickness
(mm)
Mucosal area
(mm)2
Mucosal perimeter
(mm)
Mucosal thickness
(mm)
44.861.0
55.461.9
123.7
, 0.0001
140873617797
180452611909
128.1
0.0120
73836933
70686203
95.7
1963
2661
132.6
0.0080
12460386163161
12149426385581
97.5
69396894
66266222
95.5
206650
205671
99.4
NT, Nontransgenic; T, SMP8–IGF-I transgenic; n 5 5. Comparison between male SMP8–IGF-I transgenic mice (line 23423) and nontransgenic agematched controls.
1434
Wang et al.
Figure 8. Gross anatomy
and low-power magnification sections of selected
tissues of SMP8–IGF-I
transgenic mice (TG) and
nontransgenic littermates
(NT). (a) Thickening of
muscular layer of bladder
in SMP8–IGF-I mice
(H & E stain). (b) Longitudinal growth of uterine
horns (top), with no
change in thickness of
myometrial layer (bottom). (c) (top) Gross view
of small intestine from gastric junction to cecum,
shows lengthening of small
bowel in TG mouse. (Bottom) Trichrome staining of
transverse section of intestine. Arrows indicate muscularis layer. The epithelial layer of NT mouse was
disrupted during fixation.
IGF-I Overexpression in Smooth Muscle Cells of Transgenic Mice
1435
Table III. Relative Content of DNA and RNA
(RNA/DNA) in Tissues from SMP8–IGF-I
Mice and their Nontransgenic Controls
Sample
NT
T
Bladder
Intestine
Stomach
Brain
3.0260.43
3.1560.67
0.7560.05
0.6660.10
1.1560.25
1.0260.07
0.8660.03
0.9860.18
NT, nontransgenic; T, SMP8–IGF-I transgenic; n 5 4; values are
mean6SD.
abundance in different tissue environments. Western ligand
blotting of aorta, bladder and uterus of nontransgenic and
transgenic mice showed a very similar pattern of abundance of
IGFBPs (Fig. 9). Circulating IGFBP levels as determined by
Western ligand blotting were also unchanged, as were IGFBP-4
levels measured by RIA (not shown). IGF-I has been reported
to induce IGFBP-5 gene transcription in porcine aortic SMC
(33). IGF-I also induces IGFBP-5 gene expression in skeletal
myoblasts as part of its effects on their overall program of differentiation in vitro (34). There was no significant change in
IGFBP-5 mRNA levels in bladder, aorta or stomach of SMP8–
IGF-I mice as compared to nontransgenic age- and sexmatched controls (n 5 4, not shown). Accordingly, immunoreactive IGFBP-5 levels were not increased in the bladders of
SMP8–IGF-I mice (Fig. 10). Thus, chronic IGF-I overexpression does not recapitulate some of the effects seen acutely in
Figure 10. Western blot of bladder tissue extracts from three SMP8–
IGF-I mice (TG) and three nontransgenic (NT) controls blotted with
anti–IGFBP-5 antibody. Top arrow indicates band corresponding to
IGFBP-5 (doublet may represent glycosylated and unglycosylated
forms). Lower arrow points to proteolytic fragment of IGFBP-5.
Note lower abundance of this band in TG bladder, consistent with
protection from proteolysis by local IGF-I excess. rhBP5, recombinant human IGFBP-5.
cultured SMC. However, the abundance of a 23 kD proteolytic
fragment of IGFBP-5 is clearly decreased in bladders of transgenic mice, supporting the observation that IGF-I may protect
IGFBP-5 from proteolysis (Fig. 10). There was also no significant difference in tissue IGFBP-4 mRNA or protein levels in
the transgenic mice.
IGF-I has been reported to inhibit SM-a actin gene accumulation in adult cardiac myocytes in vitro (35). We found no
effect on either SM-a actin or SM-g actin mRNA in either
bladder, uterus, intestine, or aorta of the IGF-I transgenic
mice compared to their littermates (not shown).
Discussion
Figure 9. Serum and tissue IGFBP levels in SMP8–IGF-I transgenic
mice (TG) and their nontransgenic littermates (NT). (A) 8 ml of serum from two NT or six TG mice derived from different lines; and
(B) 150 mg of total soluble protein extracts from aorta, bladder, and
uterus were analyzed by Western ligand blotting with [125 I]IGF-I.
rhBP4, recombinant human IGFBP-4.
1436
Wang et al.
SMP8–IGF-I transgenic mice show targeted overexpression of
IGF-I in SMC beds with the following hierarchy: bladder .
stomach . aorta 5 uterus . intestine. There was little or no
transgene mRNA detected in liver (the major source of endogenous expression of IGF-I) or in skeletal or cardiac muscle. Indeed, in situ hybridization demonstrates that the expression of
the transgene is confined to SMC in all tissues examined. The
transgene-derived IGF-I remains paracrine, although one line
showing high levels of overexpression had a tendency towards
higher serum IGF-I concentration, and evidence of whole
body weight gain. Allometry of SMP8–IGF-I transgenic mice
demonstrated a selective weight gain only in bladder, uterus,
stomach, intestine, and aorta. The pattern of increase in SMC
mass was specific to the tissue environment: i.e., longitudinal
growth of horns in the uterus, elongation of the intestine, concentric thickening of the muscular layers of the bladder and
stomach, increased diameter, lumen and medial layer of the
aorta.
This paper provides evidence for the first time that the phenotype of SMC can be selectively modified through a transgenic approach. Several groups have provided important insights into the properties of genes considered to be markers for
SMC (SM22a, SM-MHc), and characterized the activities of
their respective promoters during development (15, 17, 18, 36).
These studies were performed using reporter gene constructs,
and did not attempt to target a protein capable of modifying
the biological activity of SMC. Although expression of SM-a
actin has been extensively studied during embryogenesis, the
function of its promoter in vivo has not been studied. The
21074 bp 59 flanking region of mouse SM-a actin contains an
evolutionary conserved motif that represses expression in nonmyogenic fibroblast cells, a serum response element, as well as
six E-box motifs, all of which mediate high level expression in
postconfluent BC3H1 myoblasts and aortic SMC (19–21). In
addition, the first intron has been proposed to contain additional motifs important for SMC-specific transcription. The expression of SM-a actin in the mouse is detectable in SMC precursors at about E10. However, it is well recognized that SM-a
actin is transiently expressed during embryogenesis in cardiomyoblasts, myotomal myoblasts and myotubes, and only becomes restricted to SMC postnatally (10, 37). We do not know
whether this particular SM-a actin promoter fragment was
also transcriptionally active in heart and skeletal muscle during
a window of time during development. Although the type I
IGF receptor is expressed in skeletal muscle and heart at the
appropriate period of rat embryogenesis (day 13) (38), there
was no obvious phenotype in striated muscle in the transgenic
animals. Whether this is due to a lack of effect of IGF-I in
these tissues over this period, or to inactivity of the promoter
fragment remains to be studied. The pattern of expression of
the IGF-I transgene in the adult animals is consistent with the
tissue distribution of wild-type a actin gene expression after
birth. Furthermore, the SMC hyperplasia and organ remodeling were also confined to these tissues. Indeed, based on measurements of intestinal length, it appears that most effects of
IGF-I on SMC growth took place postnatally.
The role of IGF-I on prenatal growth and differentiation
has been clarified by the phenotype of mice carrying null mutations of the IGF-I, IGF-II, and IGF-I receptor genes (39–41).
IGF-I appears to play a central role in fetal growth, as the birth
weight of the IGF-I (2/2) animals was about 60% that of the
wild-type controls starting at E13.5. Many of these mice died,
and the survivors had evidence of hypoplasia of numerous tissues, including bone, muscle, and the nervous system. At birth,
SMP8–IGF-I mice did not have an obvious phenotype (i.e., intestinal lengthening), suggesting that ambient IGF-I or IGF-II
concentrations to which fetal SMC are exposed are already
quite high, and no additional prenatal growth can be derived
by further selective overexpression in these cells.
IGF-I has a prominent role in mediating the postnatal
growth-promoting effects of growth hormone (GH). Several
transgenic approaches have been used to elucidate the relative
contributions of GH and IGF-I to somatic growth (41–46).
Mice carrying copies of rat GH fused to the mouse metallothionein-1 (MT-1) gene promoter had elevated circulating
GH and IGF-I levels, and grew significantly larger than normal control mice starting at 3 wk of age. Mice carrying a MT1–IGF-I also had elevated IGF-I, but with reduced plasma GH
levels, and also exhibited longitudinal growth (47). Part of the
increased weight was due to changes in skeletal muscle and
connective tissue mass, as well as selective organomegaly of
spleen, pancreas, brain, and kidney. Data on targeted expression of IGF-I in particular cell types are more limited. Overexpression of IGF-I in cardiac myocytes has been achieved in
transgenic mice through a rat a myosin heavy chain (aMHc)–
IGF-I chimeric gene (48). These animals developed cardiac
myocyte hyperplasia and increased heart weight that manifested after z 10 wk. By contrast, overexpression of IGF-I in
skeletal myocytes through the avian skeletal a actin gene resulted in SMC hypertrophy (49). Primary cultures of myoblasts
from these transgenic mice had precocious expression of myogenin and MyoD, and of the gene products activated by these
transcription factors, suggesting that IGF-I may have accelerated skeletal muscle differentiation. SMC are not subject to
terminal differentiation, and it is perhaps not surprising that
SMC from SMP8–IGF-I mice were hyperplastic. The expression of a actin and g-enteric actin mRNAs was not significantly different in transgenics versus littermates in any of the
SMC-tissue beds, and a actin expression was not detectable in
striated muscle postnatally, indicating that IGF-I did not introduce major disruptions in their developmental program.
Whether there was a more subtle acceleration or delay of muscle development that was later compensated for is unknown.
The pattern of organ remodeling resulting from IGF-I
overexpression in SMC was particularly remarkable. Whereas
the SMC layer of bladder, aorta, and stomach grew in thickness, the small intestine and the uterine horns grew primarily
in length. The mechanisms of this intriguing phenotype are not
known. The possibility that the transgene may have been differentially expressed in the circular versus the longitudinal layers of the respective tissues deserves consideration. Uterine
mesenchymal cells remain randomly oriented during the late
fetal period. Postnatally, three layers become apparent. The
circular middle layer and the outer longitudinal layer express a
actin in a similar temporal pattern (50). Furthermore, in situ
hybridization of tissues of transgenic mice demonstrated a
fairly uniform level of expression of the transgene among the
SMC layers, indicating that the remodeling effects were not
due to targeted expression to a specific SMC tissue compartment. Similarly, studies on the distribution of IGF-I receptors
in the intestine and uterus of several species have consistently
shown higher levels in the epithelial layers, but homogeneous
distribution between SMC compartments (8, 51, 52). The IGF-I
receptor is also evenly expressed in the rat arterial media (3).
Hence, it appears unlikely that the pattern of remodeling can
be ascribed to differential transgenic IGF-I expression, or to
the distribution of the IGF-I receptor. With the possible exception of the uterus, the pattern of organ growth resembles
that observed after certain pathophysiological stimuli, or in
other nontargeted transgenic models. Transgenic mice that
ubiquitously overexpress either bovine GH (53) or IGF-I (54)
under control of the MT-1 promoter exhibit increased mucosal
thickness and an increase in intestinal length. After small
bowel resection, mice treated systemically with IGF-I analogs
that bind poorly to binding proteins for one week had a dosedependent increase in intestinal length (55). It is possible that
the drive for intestinal lengthening in the SMP8–IGF-I mice
results at least in part from diffusion of IGF-I from the SMC
compartment to the intestinal mucosa. The appearance of the
bladder in the SMP8-IGF-I transgenic mice resembles that described in models of obstructive bladder hypertrophy, where
IGF-I has been proposed to play a pathogenic role (9).
Besides modulating IGF action, the overall abundance and
distribution of IGFBPs can in turn be affected by ambient
IGF-I levels. Overexpression of IGF-I was not associated with
changes in tissue IGFBP4 or 5 mRNA or protein levels in any
of the SMC tissue beds. Similarly, the abundance of IGFBPs as
determined by Western-ligand blotting was comparable be-
IGF-I Overexpression in Smooth Muscle Cells of Transgenic Mice
1437
tween transgenic mice and their littermates. This is of interest,
as transcription of IGFBP-5 is induced by IGF-I in rat SMC in
vitro (33). This suggests that chronic overexpression of IGF-I
in vivo cannot further increase IGFBP-5 gene expression in
SMC. IGF-I has also been shown to protect IGFBP-5 from
proteolysis, although this varies depending on the cell type
(56). The bladders from the SMP8–IGF-I had a lower abundance of the 23-kD IGFBP-5 proteolytic product, indicating
that IGF-I indeed protects IGFBP-5 from cleavage by its specific protease.
We and others have reported that the expression of IGF-I
is markedly induced after balloon denudation of the rat aorta,
femoral and carotid arteries, peaking 3–7 d after the procedure
(3–5). The expression of the IGF–type I receptor is reciprocally decreased in the artery media, consistent with downregulation after ligand binding (3). Similarly, IGF-I gene expression accompanies bladder- and estrogen-induced myometrial
hyperplasia. The individual contribution of a single growth factor in a complex tissue response is hard to elucidate, because
of the participation of multiple mitogens, many of which may
partially overlap in their function. In addition to the data reported here, we have also established lines of transgenic mice
overexpressing IGFBP-4 under control of the SMP8 promoter,
and these animals have a reciprocal phenotype (Wang, J., W.
Niu, Y. Nikiforov, S. Naito, S. Chernausek, D. Witte, D. LeRoith, A. Strauch, and J. Fagin, unpublished observations).
Availability of mice with strictly paracrine alterations in the
expression of members of the IGF family of proteins will allow
a better analysis of their function under basal conditions and
after various perturbations in vivo. Use of SM-a actin 59 flanking sequences to target expression in transgenic mice should
also be valuable to explore the function of other gene products
in SMC tissues in vivo.
Acknowledgments
We are grateful to Dr. John Newman, of the University of Cincinnati
Transgenic Mouse Core Facility for generating the transgenic mice,
and to Kathy Saalfeld and Pamela Groen for assistance with the in
situ hybridization experiments.
This paper was supported by National Institutes of Health Grant
HL43802. J.A. Fagin is a recipient of an Established Investigator
Award of the American Heart Association and Bristol-Myers Squibb.
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